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Important information gaps remain on the efficacy and safety of drugs in children. Pediatric drug development encounters several ethical, practical, and scientific challenges. One barrier to the evaluation of medicines for children is a lack of innovative methodologies that have been adapted to the needs of children. This article presents our successful experience of pediatric microdose and microtracer studies using 14C-labeled probes in Europe to illustrate the strengths and limitations of these approaches. [ABSTRACT FROM AUTHOR]

Hexaminolevulinate (HAL) is a diagnostic agent that allows the visualization of tumor tissue in the bladder by fluorescence cystoscopy. It is administered intravesically via a catheter for 1 hour, followed by blue light bladder inspection to induce selective red tumor fluorescence. Hexaminolevulinate should ideally be confined to the bladder only, but it is likely that some absorption occurs during administration, and therefore the systemic bioavailability is of interest. The bioavailability of HAL was determined by intravesical and intravenous administration of [14C]-HAL hydrochloride to 8 human volunteers. To reduce the radiation dose as low as possible, the ultrasensitive analytical technique of accelerator mass spectrometry was used to measure [14C]-HAL. The bioavailability of [14C]-HAL after intravesical and intravenous administration was determined from the respective area under the curve based on total radioactivity and was determined to be 7% (range, 5%-10%; 90% confidence interval). The systemic absorption of [14C]-HAL after intravesical administration is low and supports previous clinical experience with HAL showing no systemic side effects. [ABSTRACT FROM AUTHOR]

The absolute bioavailability of nelfinavir was determined in 6 healthy volunteers following simultaneous administration of 1250 mg oral nelfinavir and an intravenous infusion of (14)C-nelfinavir mesylate on day 1 and at steady state. Nelfinavir oral bioavailability decreased from 0.88 to 0.47 over the 11-day study period. The moderate bioavailability of nelfinavir was due to significant first-pass metabolism rather than low absorption, limiting the potential of formulation improvement to decrease pill burden. Human absolute bioavailability studies with accelerator mass spectrometry microdosing, in which an intravenous microdose is given along with a conventional oral dose of the same drug, can differentiate between gastrointestinal absorption and the first-pass metabolism of new drug candidates. Accelerator mass spectrometry allowed a several thousand-fold dose reduction of (14)C-nelfinavir relative to that required for liquid scintillation counting. Accelerator mass spectrometry microdosing reduces potential safety issues around dosing radioactivity to humans and prevents the need to formulate high intravenous doses. [ABSTRACT FROM AUTHOR]

Importance: Suicide risk is a global public health crisis, with suicide ranking as a consistent leading cause of death among adults in the US. Autistic individuals and transgender or gender-nonconforming (TGNC) individuals represent populations with notably elevated rates of suicidal thoughts and behaviors (STBs)., Objective: To characterize suicidal thoughts and behaviors among TGNC and autistic individuals, using a large, nationally representative sample., Design, Setting, and Participants: This study is a secondary analysis of cross-sectional data from students at colleges and universities throughout the US who participated in the American College Health Association National College Health Assessment from 2019 to 2023., Exposures: Autistic and TGNC identities were self-reported by participants., Main Outcomes and Measures: The frequency of intersectionality of autism and TGNC identities and whether those who had intersectional marginalized identities had increased likelihood of STBs were examined. STBs were self-reported by participants. A series of moderated regression analyses were performed to examine how the interaction between autism and possessing a marginalized gender identity (ie, TGNC status) was associated with STBs., Results: The sample included 41 507 college students with a mean (SD) age of 23.35 (6.83) years. A total of 2410 participants (5.81%) identified as being TGNC. Overall, 326 TGNC participants (13.53%) also identified as autistic, whereas 625 of those who identified as cisgender (1.58%) also identified as autistic. Gender identity and autism were associated with greater odds of STBs. For suicidal ideation, gender identity had an odds ratio (OR) of 3.34 (95% CI, 2.99-3.73), and autism had an OR of 2.06 (95% CI, 1.76-2.42). For suicide attempts, gender identity had an OR of 2.74 (95% CI, 2.13-3.52), and autism had an OR of 2.39 (95% CI, 1.62-3.52). A significant interaction existed for attempts (OR, 0.51; 95% CI, 0.27-0.97); nonautistic cisgender individuals had the lowest attempt rate., Conclusions and Relevance: This cross-sectional study addresses the dearth of information on how intersectionality in gender and autism status impacts the risk of STBs, and the results confirm the elevated risk of STBs among TGNC and autistic populations. Interventions are needed to support college students with these identities.

Aims: Drug disposition in children may vary from adults due to age-related variation in drug metabolism. Microdose studies present an innovation to study pharmacokinetics (PK) in paediatrics; however, they should be used only when the PK is dose linear. We aimed to assess dose linearity of a [ 14 C]midazolam microdose, by comparing the PK of an intravenous (IV) microtracer (a microdose given simultaneously with a therapeutic midazolam dose), with the PK of a single isolated microdose., Methods: Preterm to 2-year-old infants admitted to the intensive care unit received [ 14 C]midazolam IV as a microtracer or microdose, followed by dense blood sampling up to 36 hours. Plasma concentrations of [ 14 C]midazolam and [ 14 C]1-hydroxy-midazolam were determined by accelerator mass spectrometry. Noncompartmental PK analysis was performed and a population PK model was developed., Results: Of 15 infants (median gestational age 39.4 [range 23.9-41.4] weeks, postnatal age 11.4 [0.6-49.1] weeks), 6 received a microtracer and 9 a microdose of [ 14 C]midazolam (111 Bq kg -1 ; 37.6 ng kg -1 ). In a 2-compartment PK model, bodyweight was the most significant covariate for volume of distribution. There was no statistically significant difference in any PK parameter between the microdose and microtracer, nor in the area under curve ratio [ 14 C]1-OH-midazolam/[ 14 C]midazolam, showing the PK of midazolam to be linear within the range of the therapeutic and microdoses., Conclusion: Our data support the dose linearity of the PK of an IV [ 14 C]midazolam microdose in children. Hence, a [ 14 C]midazolam microdosing approach may be used as an alternative to a therapeutic dose of midazolam to study developmental changes in hepatic CYP3A activity in young children., (© 2019 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The Prestwick library was screened for antibacterial activity or "antibiotic resistance breaker" (ARB) potential against four species of Gram-negative pathogens. Discounting known antibacterials, the screen identified very few ARB hits, which were strain/drug specific. These ARB hits included antimetabolites (zidovudine, floxuridine, didanosine, and gemcitabine), anthracyclines (daunorubicin, mitoxantrone, and epirubicin), and psychoactive drugs (gabapentin, fluspirilene, and oxethazaine). These findings suggest that there are few approved drugs that could be directly repositioned as adjunct antibacterials, and these will need robust testing to validate efficacy., (© Crown copyright 2019.)

Accelerator mass spectrometry (AMS) is an ultrasensitive technique to detect radiolabeled compounds. We administered a microdose (100 µg) of (14)C-labeled zidovudine (ZDV) with or without a standard unlabeled dose (300 mg) to healthy volunteers. Intracellular ZDV-triphosphate (ZDV-TP) concentration was measured using AMS and liquid chromatography-tandem mass spectrometry (LC/MS/MS). AMS analysis yielded excellent concordance with LC/MS/MS and was 30,000-fold more sensitive. The kinetics of intracellular ZDV-TP formation changed several-fold over the dose range studied (100 µg-300 mg). AMS holds promise as a tool for quantifying intracellular drug metabolites and other biomediators in vivo.

The background to human microdosing or Phase 0 studies is reviewed, focusing particularly on the information that such studies can provide in the context of exploratory clinical development. Examples are provided of the microdose-validation studies known as the Consortium for Resourcing and Evaluating AMS Microdosing trial and EU Microdosing AMS Partnership Programme, which demonstrated that there was good dose proportionality between microdose and pharmacological dose pharmacokinetics. When microdosing was applied to ten development drugs, it was found that all ten molecules showed dose proportionality between the microdose and the pharmacological dose. The majority of microdose studies have used accelerator mass spectrometry (AMS) analysis and only these studies that are considered here; AMS provides information on all metabolites, even if these are minor. There is now sufficient scientific data to justify microdose studies being routinely conducted as part of the drug-development process.

IDX899 and IDX989 are new non-nucleoside reverse-transcriptase inhibitors (NNRTIs) that exhibit potent inhibition of HIV-1 replication, including NNRTI-resistant mutants. This microdose study investigates the pharmacokinetics and determined oral bioavailability. For each compound, 4 healthy male subjects are randomized to receive via a crossover design a single 100-microg oral and intravenous dose together with 100 nCi of [(14)C]-labeled drug. Plasma and urine samples are obtained over a period of 168 hours postdose and analyzed for total, unchanged drug and major metabolites using an accelerator mass spectrometry method. Based on total radioactivity, oral absorption is near complete. For the parent drug, mean absolute bioavailability is 61% and 65% for IDX899 and IDX989, respectively. Both compounds are extensively metabolized especially after oral dosing. Observed terminal phase half-lives after oral and intravenous doses range from 4 to 10 hours and are comparable for the 2 compounds. Urine excretion of radioactivity for both compounds is less than 10%. These data show for the first time that IDX899 and IDX989 possess favorable pharmacokinetic properties in humans, including high mean absolute bioavailability and long half-life. IDX899 has been selected based on these initial pharmacokinetic assessments and other criteria as the candidate for further clinical development.

Aims: To evaluate the pharmacokinetics (PK) of five H(1) receptor antagonists in human volunteers after a single oral and intravenous (i.v.) microdose (0.1 mg)., Methods: Five H(1) receptor antagonists, namely NBI-1, NBI-2, NBI-3, NBI-4 and diphenhydramine, were administered to human volunteers as a single 0.1-mg oral and i.v. dose. Blood samples were collected up to 48 h, and the parent compound in the plasma extract was quantified by high-performance liquid chromatography and accelerator mass spectroscopy., Results: The median clearance (CL), apparent volume of distribution (V(d)) and apparent terminal elimination half-life (t(1/2)) of diphenhydramine after an i.v. microdose were 24.7 l h(-1), 302 l and 9.3 h, and the oral C(max) and AUC(0-infinity) were 0.195 ng ml(-1) and 1.52 ng h ml(-1), respectively. These data were consistent with previously published diphenhydramine data at 500 times the microdose. The rank order of oral bioavailability of the five compounds was as follows: NBI-2 > NBI-1 > NBI-3 > diphenhydramine > NBI-4, whereas the rank order for CL was NBI-4 > diphenhydramine > NBI-1 > NBI-3 > NBI-2., Conclusions: Human microdosing provided estimates of clinical PK of four structurally related compounds, which were deemed useful for compound selection.

Background: Microdosing studies (human Phase 0) are used to select drug candidates for Phase I clinical trials on the basis of their pharmacokinetic properties, using subpharmacologic doses (maximum 100 microg). There are questions as to whether pharmacokinetic data obtained at these low doses will predict those at the clinically relevant dose., Objective: To review the current literature on microdosing and assess how well microdose data have predicted the pharmacokinetics obtained at a therapeutic dose., Methods: All data published in the peer reviewed literature comparing pharmacokinetics at a microdose with a therapeutic dose were reviewed, excluding those studies aimed at imaging., Conclusions: Of the 18 drugs reported, 15 demonstrated linear pharmacokinetics within a factor of 2 between a microdose and a therapeutic dose. Therefore, data that support the utility of microdosing are beginning to emerge.

Ixabepilone (BMS-247550) is a semi-synthetic, microtubule stabilizing epothilone B analogue which is more potent than taxanes and has displayed activity in taxane-resistant patients. The human plasma pharmacokinetics of ixabepilone have been described. However, the excretory pathways and contribution of metabolism to ixabepilone elimination have not been determined. To investigate the elimination pathways of ixabepilone we initiated a mass balance study in cancer patients. Due to autoradiolysis, ixabepilone proved to be very unstable when labeled with conventional [14C]-levels (100 microCi in a typical human radio-tracer study). This necessitated the use of much lower levels of [14C]-labeling and an ultra-sensitive detection method, Accelerator Mass Spectrometry (AMS). Eight patients with advanced cancer (3 males, 5 females; median age 54.5 y; performance status 0-2) received an intravenous dose of 70 mg, 80 nCi of [14C]ixabepilone over 3 h. Plasma, urine and faeces were collected up to 7 days after administration and total radioactivity (TRA) was determined using AMS. Ixabepilone in plasma and urine was quantitated using a validated LC-MS/MS method. Mean recovery of ixabepilone-derived radioactivity was 77.3% of dose. Fecal excretion was 52.2% and urinary excretion was 25.1%. Only a minor part of TRA is accounted for by unchanged ixabepilone in both plasma and urine, which indicates that metabolism is a major elimination mechanism for this drug. Future studies should focus on structural elucidation of ixabepilone metabolites and characterization of their activities.

Objectives: A volunteer trial was performed to compare the pharmacokinetics of 5 drugs--warfarin, ZK253 (Schering), diazepam, midazolam, and erythromycin--when administered at a microdose or pharmacologic dose. Each compound was chosen to represent a situation in which prediction of pharmacokinetics from either animal or in vitro studies (or both) was or is likely to be problematic., Methods: In a crossover design volunteers received (1) 1 of the 5 compounds as a microdose labeled with radioactive carbon (carbon 14) (100 microg), (2) the corresponding (14)C-labeled therapeutic dose on a separate occasion, and (3) simultaneous administration of an intravenous (14)C-labeled microdose and an oral therapeutic dose for ZK253, midazolam, and erythromycin. Analysis of (14)C-labeled drugs in plasma was done by use of HPLC followed by accelerator mass spectrometry. Liquid chromatography-tandem mass spectrometry was used to measure plasma concentrations of ZK253, midazolam, and erythromycin at therapeutic concentrations, whereas HPLC-accelerator mass spectrometry was used to measure warfarin and diazepam concentrations., Results: Good concordance between microdose and therapeutic dose pharmacokinetics was observed for diazepam (half-life [t((1/2))] of 45.1 hours, clearance [CL] of 1.38 L/h, and volume of distribution [V] of 90.1 L for 100 microg and t((1/2)) of 35.7 hours, CL of 1.3 L/h, and V of 123 L for 10 mg), midazolam (t((1/2)) of 4.87 hours, CL of 21.2 L/h, V of 145 L, and oral bioavailability [F] of 0.23 for 100 microg and t((1/2)) of 3.31 hours, CL of 20.4 L/h, V of 75 L, and F of 0.22 for 7.5 mg), and development compound ZK253 (F = <1% for both 100 microg and 50 mg). For warfarin, clearance was reasonably well predicted (0.17 L/h for 100 microg and 0.26 L/h for 5 mg), but the discrepancy observed in distribution (67 L for 100 microg and 17.9 L for 5 mg) was probably a result of high-affinity, low-capacity tissue binding. The oral microdose of erythromycin failed to provide detectable plasma levels as a result of possible acid lability in the stomach. Absolute bioavailability for the 3 compounds examined yielded excellent concordance with data from the literature or data generated in house., Conclusion: Overall, when used appropriately, microdosing offers the potential to aid in early drug candidate selection.

Although 14C-labelling has been routinely used for small molecules, this technique is not routinely applied to therapeutic proteins due to difficulties of incorporating the label into the protein to a sufficiently high specific activity. An analytical method known as accelerator mass spectrometry (AMS) offers an extremely sensitive method of 14C quantification, thereby enabling (14)C-labeling methods to be applied to therapeutic protein detection. The therapeutic protein CAT-192 (metelimumab), a human anti-TGFss1 monocloncal antibody was manufactured in the presence of 14C-precursors resulting in a low specific activity product (1.4% 14C incorporation). [14C]-CAT-192 was administered to rats (1mg/kg and 222, 22 and 2.2 dpm/kg) and serum samples were collected. 14C in serum samples from the 2.2 dpm dosing was not detectable but samples from the 22 and 2220 dpm doses were measured by AMS and by ELISA for comparison. By both ELISA and AMS bioassay, the half-lives approximated 140 h (S.E.M. 15 h). The estimates of clearance were also comparable, 7.3 and 4.6 x 10(-4)ml/h/g (S.E.M. 6.6 and 5.1 x 10(-5)) for ELISA and AMS, respectively. The estimated limit of quantification (LOQ) was approximately 1 ng/ml, about 15 times lower than the ELISA LOQ of 15.6 ng/ml.

Absolute bioavailability studies in humans are not routinely performed as part of the drug registration process. They tend to be reasonably demanding, not least because toxicology data are required to support intravenous administration of a drug. Moreover, the classical crossover design of an absolute bioavailability study can suffer from artefacts caused by concentration-dependent pharmacokinetics. Many of the problems associated with absolute bioavailability studies can be alleviated using isotopically labelled drugs. Stable isotopes have been used in the performance of absolute bioavailability studies in humans for > 30 years. More recently, the advantages of using radiolabelled drugs have been expanded by using the ultrasensitive technology of accelerator mass spectrometry. Isotopic labelling not only allows for the accurate and efficient determination of absolute bioavailability, but can also provide information on first-pass effects and other pharmacokinetic parameters.

There is an increasing recognition within the pharmaceutical industry of the importance of the ADME studies in drug registration. Consequently, there has been a drive in recent times to conduct the ADME studies as early as possible in the development programme. There are, however, regulatory barriers, particularly in the administration of radiotracers to human volunteers, which place limitations on the timing of the ADME studies. Accelerator mass spectrometry (AMS), a technology new to the pharmaceutical industry, is an ultrasensitive technique for measuring tracers such as (14)C. Using AMS, it is possible to lower the radioactive dose administered to humans to a point where many regulatory authorities consider it insignificant. With the removal of the regulatory hurdles, ADME data can be obtained much earlier in the development process. Tracers such as (14)C can be administered in minute amounts in the first in man studies (Phase I), or even in a preregulatory study known as microdosing (or human Phase 0). AMS also enables other studies such as absolute bioavailability to be conducted earlier if required.

Epidemiological evidence shows high red meat consumption to increase the risk of colorectal cancer, while the consumption of fruit and vegetables has been shown to be protective. Many genes have been identified that encode for enzymes involved in the metabolism of dietary carcinogens or anti-carcinogens. A study of 500 incident colorectal cancer cases and population controls, matched for age, sex and general practitioner, was conducted in the United Kingdom to investigate whether 6 such genes (CYP1A1, GSTT1, GSTM1, GSTP1, EPHX1 and NQO1) modify the relationship between diet and disease risk. Usual diet was estimated using a detailed questionnaire administered by interview. Fruit and vegetable consumption were both found to protect against colorectal cancer, while overall meat and red meat consumption were found to increase risk. There was some evidence of interaction between GSTT1 and vegetable consumption (p=0.006, not adjusted for multiple tests) but no evidence of interaction with GSTM1. The protective effect of vegetables was only seen in those with deficient or intermediate GSTT1 predicted phenotype [OR 0.3, 95% confidence interval (0.1, 0.6), and OR 0.6 (0.4, 0.96), OR 1.4 (0.3, 2.4) for those with fast phenotype], and a similar result was observed for cruciferous vegetables. There was also weak evidence of interaction between red meat intake and GSTT1 (p=0.06), GSTP1 (p=0.16, with p=0.02 after adjustment for potential confounders) and NQO1 predicted phenotype (p=0.01). Because of the multiple hypotheses tested in our study, these findings require independent confirmation., (Copyright 2004 Wiley-Liss, Inc.)

The levels of aflatoxin B(1)-DNA and aflatoxin B(1)-albumin adducts were investigated by accelerator mass spectrometry (AMS) in humans and rats following exposure to a known, dietary relevant amount of carbon-14 labeled aflatoxin B(1) ([(14)C]AFB(1)). The aims of the study were to: (a) investigate the dose-dependent formation of DNA and protein adducts at very low doses of AFB(1) (0.16 ng/kg-12.3 microg/kg) in the rat; (b) measure the levels of AFB(1)-albumin and AFB(1)-DNA adducts at known, relevant exposures in humans (c) study rat to human extrapolations of AFB(1)-albumin and DNA adduct levels. The results in the rat showed that both AFB(1)-albumin adduct and AFB(1)-DNA adduct formation were linear over this wide dose range. The order of adduct formation within the tissues studied was liver>kidney>colon>lung=spleen. Consenting volunteers received 1 microg ( approximately 15 ng/kg) of [(14)C]AFB(1) in a capsule approximately approximately 3.5-7 h prior to undergoing colon surgery. The mean level of human AFB(1)-albumin adducts was 38.8+/-19.55 pg [(14)C]AFB(1)/mg albumin/microg AFB(1)/kg body weight (b.w.), which was not statistically different to the equivalent dose in the rat (15 ng/kg) 42.29+/-7.13 pg [(14)C]AFB(1)/mg albumin/microg AFB(1)/kg b.w. There was evidence to suggest the formation of AFB(1)-DNA adducts in the human colon at very low doses. Comparison of the linear regressions of hepatic AFB(1)-DNA adduct and AFB(1)-albumin adduct levels in rat found them to be statistically similar suggesting that the level of AFB(1)-albumin adducts are useful biomarkers for AFB(1) dosimetry and may reflect the DNA adduct levels in the target tissue. [(14)C]AFB(1)-DNA and [(14)C]AFB(1)-albumin adducts were hydrolysed and analysed by HPLC to confirm that the [(14)C] measured by AMS was derived from the expected [(14)C]AFB(1) adducts.

Accelerator mass spectrometry (AMS) is an extremely sensitive nuclear physics technique developed in the mid-70's for radiocarbon dating of historical artefacts. The technique centres round the use of a tandem Van de Graaff accelerator to generate the potential energy to permit separation of elemental isotopes at the single atom level. AMS was first used in the early 90's for the analysis of biological samples containing enriched 14C for toxicology and cancer research. Since that time biomedical AMS has been used in the study of (1) metabolism of xenobiotics in animals and humans (2) pathways of drug metabolism (3) biomarkers (4) metabolism of endogenous molecules including vitamins (5) DNA and protein binding studies and (6) clinical diagnosis. A new drug development concept which relies on the ultrasensitivity of AMS known as human microdosing (Phase 0) is being used to obtain early human metabolism information of candidate drugs arising out of discovery. These various aspects of AMS are reviewed in this article and a perspective on future applications of AMS provided.

The formation of DNA adducts in human HepG2 cells and human hepatocytes exposed to 14C-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined using Accelerator Mass Spectrometry (AMS). PhIP generated DNA adducts in a linear dose-dependent manner between 100 pM and 20 micro M. Co-treatment with the dietary isothiocyanate, sulforaphane (SFN, 1-10 micro M), or the flavonoid, quercetin (5-20 micro M), significantly reduced the level of PhIP-DNA adducts in a dose-dependent manner. The degree of protection was dependent on PhIP concentration, i.e. after 100 pM PhIP exposure, SFN or quercetin reduced adduct levels to below the limit of detection (0.15 amol PhIP/ micro g DNA) but at higher PhIP exposure (10 nM and 1 micro M), the protection was 60 and 10%, respectively. The involvement of phase I, phase II and DNA repair enzymes in this protection against PhIP-DNA adduct formation was investigated using real-time RT-PCR and enzyme activity assays. In intact HepG2 cells, quercetin inhibited cytochrome P450 (CYP)1A2, the main phase I enzyme responsible for PhIP bioactivation. In contrast, SFN induced phase II detoxification enzymes, UDP-glucuronosyltransferase 1A1 and glutathione S-transferase A1 mRNA expression. SFN and quercetin showed no effect on DNA repair, neither in terms of the level of PhIP-DNA adducts, when cells were treated with phytochemicals after the carcinogen exposure, nor the regulation of mRNA expression of two DNA repair enzymes, apurinic endonuclease and DNA polymerase beta. This study indicates that dietary isothiocyanates and flavonoids modulate phase I and phase II enzyme expression, hence increasing the rate of detoxification of the dietary carcinogen PhIP in human HepG2 cells but do not affect the rate of PhIP-DNA adduct repair. The formation of PhIP-DNA adducts in human hepatocytes was also dose-dependent with PhIP-concentration and the levels of protection by SFN or quercetin were up to 60% after 10 nM PhIP treatment, but showed large inter-individual variation with no observed protection in some individuals.

This study was aimed to establish whether tamoxifen binds irreversibly to uterine DNA when given to women. Patients were given a single therapeutic dose of [(14)C]tamoxifen citrate orally (20 mg, 0.37 or 1.85 MBq) approximately 18 h prior to hysterectomy or breast surgery. Nonmalignant uterine tissue was separated into myometrium and endometrium. DNA and protein were isolated and bound radiolabel determined by the sensitive technique of accelerator mass spectrometry. Levels of irreversible DNA binding of tamoxifen in the endometrium of treated patients were 237 +/- 77 adducts/10(12) nucleotides (mean +/- SE, n = 10). In myometrial tissues, a similar extent of DNA binding was detected (492 +/- 112 adducts/10(12) nucleotides). Binding of tamoxifen to endometrial and myometrial proteins was 10 +/- 3 and 20 +/- 4 fmol/mg, respectively. In breast tissue, sufficient DNA could not be extracted but protein binding was an order of magnitude higher than that seen with endometrial proteins (358 +/- 81 fmol/mg). These results demonstrate that after oral administration, tamoxifen forms adducts in human uterine DNA but at low numbers relative to those previously reported in women after long-term tamoxifen treatment where levels, when detected, ranged from 15000 to 130000 adducts/10(12) nucleotides. Our findings support the hypothesis that the low level of DNA adducts in human uterus is unlikely to be involved with endometrial cancer development.

Testing the safety and efficacy of a successful human medicine involves many laboratory animals, which can sometimes be subjected to considerable suffering and distress. Also, it is necessary to extrapolate from the test species to humans. UK and European legislation requires that Replacement, Reduction and Refinement of animal procedures (the Three Rs) are implemented wherever possible. Over the last decade, there has been substantial progress with applying in vitro and in silico methods to both drug efficacy and safety testing. This paper is a report of the discussions and recommendations arising from a workshop on the role that might be played by human volunteer studies in the very early stages of drug development. The workshop was organised in November, 2001 by Volunteers in Research and Testing, a group of individuals in the UK which launched an initiative in 1994 to identify where and how human volunteers can participate safely in biomedical studies to replace laboratory animals. It was considered that conducting pre-Phase I very low dose human studies (sub-toxic and below the dose threshold for measurable pharmacological or clinical activity) could enable drug candidates to be assessed earlier for in vivo human pharmacokinetics and metabolism. Moreover, accelerator mass spectrometry (AMS), nuclear magnetic resonance (NMR) spectroscopy and positron emission tomography (PET) are potentially useful spectrometric and imaging methods that can be used in conjunction with such human studies. Some, limited animal tests would still be required before pre-Phase I microdose studies, to take account of the potential risk posed by completely novel chemicals. The workshop recommended that very early volunteer studies using microdoses should be introduced into the drug development process in a way that does not compromise volunteer safety or the scientific quality of the resulting safety data. This should improve the selection of drug candidates and also reduce the likelihood of later candidate failure, by providing in vivo human ADME data, especially for pharmacokinetics and metabolism, at an earlier stage in drug development than is currently the case.

The process of early clinical drug development has changed little over the past 20 years despite an up to 40% failure rate associated with inappropriate drug metabolism and pharmacokinetics of candidate molecules. A new method of obtaining human metabolism data known as microdosing has been developed which will permit smarter candidate selection by taking investigational drugs into humans earlier. Microdosing depends on the availability of two ultrasensitive 'big-physics' techniques: positron emission tomography (PET) can provide pharmacodynamic information, whereas accelerator mass spectrometry (AMS) provides pharmacokinetic information. Microdosing allows safer human studies as well as reducing the use of animals in preclinical toxicology.

Fast N-acetyltransferase 2 (NAT2) acetylators may be at increased risk of colorectal cancer through the activation of carcinogenic heterocyclic amines (HA), which are produced by meat cooked at high temperatures and are found in cigarette smoke. A study of 500 incident colorectal cancer cases and population controls, matched for age, sex and general practitioner, was conducted in the UK to investigate this hypothesis. Usual meat intake and lifetime smoking habits were estimated using a detailed questionnaire administered by interview. Subjects also indicated how well cooked they ate their meat. Subjects were classified as fast or slow NAT2 acetylators on the basis of NAT2 genotype. Complete genotype data were available on 433 matched pairs. The risk of colorectal cancer showed a steady increase with meat intake, rising to an odds ratio of 1.51 [95% confidence interval (1.03, 2.23)] for the highest versus the lowest quartile, after adjustment for total energy intake, and this was even more pronounced for red meat [odds ratio 1.97 (1.30, 2.98)]. However, this effect was not influenced by the preference for well-done meat. Smoking was also associated with an increased risk [odds ratio 1.47 (1.10, 1.98) for ever- versus never-smokers]. In both cases and controls approximately 40% of subjects were classified as fast acetylators, and the risks associated with (red) meat intake and smoking did not vary with NAT2 status. This study provides no support for the hypothesis that fast NAT2 acetylators are at increased risk of colorectal cancer, even if exposed to high levels of HA from well-cooked meat or smoking.

Aims: Several single nucleotide polymorphisms (SNPs) of the cytochrome P450 enzyme 1A2 gene (CYP1A2) have been reported. Here, frequencies, linkage disequilibrium and phenotypic consequences of six SNPs are described., Methods: From genomic DNA, 114 British Caucasians (49 colorectal cancer cases and 65 controls) were genotyped for the CYP1A2 polymorphisms -3858G-->A (allele CYP1A2*1C), -2464T-->delT (CYP1A2*1D), -740T-->G (CYP1A2*1E and *1G), -164A-->C (CYP1A2*1F), 63C-->G (CYP1A2*2), and 1545T-->C (alleles CYP1A2*1B, *1G, *1H and *3), using polymerase chain reaction-restriction fragment length polymorphism assays. All patients and controls were phenotyped for CYP1A2 by h.p.l.c. analysis of urinary caffeine metabolites., Results: In 114 samples, the most frequent CYP1A2 SNPs were 1545T-->C (38.2% of tested chromosomes), -164A-->C (CYP1A2*1F, 33.3%) and -2464T-->delT (CYP1A2*1D, 4.82%). The SNPs were in linkage disequilibrium: the most frequent constellations were found to be -3858G/-2464T/-740T/-164A/63C/1545T (61.8%), -3858G/-2464T/-740T/-164C/63C/1545C (33.3%), and -3858G/-2464delT/-740T/-164A/63C/1545C (3.51%), with no significant frequency differences between cases and controls. In the phenotype analysis, lower caffeine metabolic ratios were detected in cases than in controls. This was significant in smokers (n = 14, P = 0.020), and in a subgroup of 15 matched case-control pairs (P = 0.007), but it was not significant in nonsmokers (n = 100, P = 0.39). There was no detectable association between CYP1A2 genotype and caffeine phenotype., Conclusions: (i) CYP1A2 polymorphisms are in linkage disequilibrium. Therefore, only -164A-->C (CYP1A2*1F) and -2464T-->delT (CYP1A2*1D) need to be analysed in the routine assessment of CYP1A2 genotype; (ii) in vivo CYP1A2 activity is lower in colorectal cancer patients than in controls, and (iii) CYP1A2 genotype had no effect on phenotype (based on the caffeine metabolite ratio). However, this remains to be confirmed in a larger study.

Colorectal cancer is one of the most significant causes of cancer death. A genetic model for colorectal cancer has been proposed in which the sequential accumulation of mutations in specific genes, including adenomatous polyposis coli (APC), Kirsten-ras (K-ras), and p53, drives the transition from healthy colonic epithelia through increasingly dysplastic adenoma to colorectal cancer. We have characterized tumor mutation spectra in a large cohort of colorectal cancer patients. In marked contrast to the predictions of the sequential model of mutation accumulation, only 6.6% of tumors were found to contain mutations in APC, K-ras, and p53, with 38.7% of tumors containing mutations in only one of these genes. The most common combination of mutations was p53 and APC (27.1%), whereas mutations in both p53 and K-ras were extremely rare. Statistical analysis (two-sided Fisher's exact test) confirmed that mutations in K-ras and p53 co-occurred less frequently than expected by chance (P < 0.01, Fisher's exact test). This finding suggests that these mutations lie on alternate pathways of colorectal tumor development. The heterogeneous pattern of tumor mutations in our patient cohort suggests that multiple alternative genetic pathways to colorectal cancer exist and that the widely accepted genetic model of cancer development is not representative of the majority of colorectal tumors.

Accelerator mass spectrometry (AMS) has been used in a human mass balance and metabolism study to analyze samples taken from four healthy male adult subjects administered nanoCurie doses of the farnesyl transferase inhibitor 14C-labeled (R)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone ([14C]R115777). Plasma, urine, and feces samples were collected at fixed timepoints after oral administration of 50 mg [14C]R115777 (25.4 Bq/mg or 687 pCi/mg i.e., equivalent to 76.257 x 10(3) dpm) per subject. AMS analysis showed that drug-related (14)C was present in the plasma samples with C(max) values ranging from 1.6055 to 2.9074 dpm/ml (1.0525-1.9047 microg/ml) at t(max) = 2 to 3 h. The C(max) values for acetonitrile extracts of plasma samples ranged from 0.3724 to 0.7490 dpm/ml in the four male subjects. Drug-related 14C was eliminated from the body both in the urine and the feces, with a mean total recovery of 79.8 +/- 12.9% in the feces and 13.7 +/- 6.2% in the urine. The majority of drug-related radioactivity in urine and feces was excreted within the first 48 h. High-performance liquid chromatography (HPLC)-AMS profiles were generated from radioactive parent drug plus metabolites from pooled diluted urine, plasma, and methanolic feces extracts and matched to retention times of synthetic reference substances, postulated as metabolites. All HPLC separations used no more than 5 dpm injected on-column. The radioactive metabolite profiles obtained compared well with those obtained using liquid chromatography/tandem mass spectometry. This study demonstrates the use of AMS in a human phase I study in which the administered radioactive dose was at least 1000-fold lower than that used for conventional radioactive studies.

Daflon 500 mg, is a micronized purified flavonoid fraction, containing 90% w/w diosmin and 10% w/w of flavonoids expressed as hesperidin, used clinically in the treatment of chronic venous insufficiency and hemorrhoidal disease. This study was designed to investigate the influence of particle size on the overall absorption of diosmin after oral administration of micronized (mean particle size = 1.79 microm, with 80% of particles having a size lower than 3.45 microm) and nonmicronized diosmin (mean particle size = 36.5 microm, with 80% of particles comprised between 19.9 and 159 microm). In a double blinded, cross-over study design, 500 mg tablets containing trace amounts (approximately 25 nCi) of (14)C-diosmin were administered to 12 healthy male volunteers as a single oral dose. Accelerator mass spectrometry and liquid scintillation counting were used for the measurement of (14)C-diosmin in urine and feces. Absorption of (14)C-diosmin from the gastrointestinal tract, measured by the urinary excretion of total radioactivity, was significantly improved with the micronized (57.9 +/- 20.2%) compared with the nonmicronized material (32.7 +/- 18.8%). Statistical comparison of the urinary excretion of the two pharmaceutical formulations showed this difference to be highly significant (p = 0.0004, analysis of variance). The overall excretion of the radiolabeled dose was 100% with mean +/- SD of 109 +/- 23% and 113 +/- 20% for the micronized and nonmicronized forms, respectively. The results of this study show: 1. the impact of a reduction of particle size on the extent of absorption of diosmin, giving a pharmacokinetic explanation to the better clinical efficacy observed with the micronized formulation, and 2. the use of accelerator mass spectrometry in conjunction with liquid scintillation counting in measurement of bioavailability in a human cross-over study comparing two drug formulations containing trace amounts of radioactivity., (Copyright 2002 Wiley-Liss, Inc.)

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20microg of 14C PhIP (182kBq, specific activity 2.05GBq/mmol) or 5microg of 14C B[a]P (36kBq, specific activity 1.81GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both 14C PhIP and 14C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22-477.35 and 6.61-208. 38 adducts/10(12) nucleotides following administration of 14C PhIP and 14C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.

A comparison has been made between accelerator mass spectrometry (AMS) analysis and liquid scintillation counting (LSC) of plasma, urine and faecal samples containing 14C-labelled drugs. In an in vitro study in which human plasma was spiked (the term spiked is used in Section 2.6) with 14C-Fluconazole (14C-FL) over a concentration range of 0.1-2.5 dpm/ml, a correlation coefficient of 0.999 was determined for AMS analysis versus extrapolated LSC data. No significant day to day (or inter-day)variation was seen (P < 0.05 by ANOVA). Coefficients of variation for these analyses ranged from 2.68 to 6.50%. In vivo studies in which rats were given a high (11.5 microCi/kg) or low (18.1 nCi/kg) radioactive dose (to model an exposure of 0.9 microSievert to man) of 14C-Fluticasone propionate(14C-FP) showed that there was also a good correspondence between AMS and LSC data. A mass balance study in a single the faeces by 96 h; less than 1% of the administered dose was excreted in the urine. The limit of reliable measurement of drug related material, above background concentrations, by AMS analysis in this study was approximately 0.1 dpm/ml for plasma, 0.01 dpm/ml for urine without any sample extraction or concentration and 0.01 dpm/ml for faecal extracts. The data reported here demonstrate that AMS is an ultrasensitive and reliable method for analysing 14C-labelled drugs in human and animal body fluids.

Accelerator mass spectrometry (AMS) permits the measurement of elemental isotopes at the individual atom level. The main application of AMS in drug discovery and development will be in the analysis of 14-carbon (14C). The principle behind AMS is the separation of individual positively charged atoms through mass, charge and momentum differences. In order to obtain the high-energy charge state required for separation, negative atoms are accelerated through a high voltage field (up to 10 million volts) generated by a tandem Van de Graaff accelerator. In the middle of the accelerator, the outer valency electrons are stripped from the atom and the resulting charged species are separated and counted. For 14C, AMS counts the number of individual atoms rather than measuring radioactive decays. The result is that AMS is up to one million times more sensitive than decay counting. Radioactivity levels as low 0.0001 dpm can be detected using AMS. The exquisite sensitivity of AMS analysis means that much lower amounts of 14C can be used than for conventional counting methods. This makes it easier to use 14C for in vitro, preclinical and clinical research programmes. As 14C poses both a biological and environmental hazard, AMS permits much lower doses to be used. Human drug mass balance studies have been conducted with doses of 50 nanoCuries and below. Radioactive HPLC metabolite profiles of plasma extracts from subjects given nanoCurie doses of 14C-labelled drug have been obtained by injecting as little as 0.25 dpm onto an HPLC column. In studies of biologics, biosynthetically 14C-labelled recombinant protein has been produced with a specific radioactivity sufficient to conduct human clinical studies with AMS analysis. For one human recombinant protein an increase in sensitivity of 2,000-fold over ELISA was obtained with AMS measurement. AMS is an enabling technology that should prove of value in increasing human and environmental safety as well as allowing new research directions to be followed.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.

MeIQx and PhIP are putative carcinogenic heterocyclic amines formed during the cooking of meat and fish. Using accelerator mass spectrometry, we have investigated the metabolism and macromolecule binding of 14C-labelled MeIQx and PhIP in human cancer patients compared to the rat. Following oral administration of MeIQx and PhIP, more DNA adducts were formed in human colon tissue compared with rats. Differences were also observed between rats and humans in the metabolite profile and urine excretion for these compounds. These results suggest humans metabolise heterocyclic amines differently to laboratory rodents and question their use as models of human risk.

[2-14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was administered orally (304 ng/kg body-weight dose based upon an average 70-kg-body-weight subject) to 5 human colon-cancer patients (58 to 84 years old), as well as to F344 rats and B6C3F1 mice. Colon tissue was collected from the human subjects at surgery and from the rodents 3.5 to 6 hr after administration. Colon DNA-adduct levels and tissue available doses were measured by accelerator mass spectrometry (AMS). The mean levels of MeIQx in the histologically normal colon tissue were not different among the human (97 +/- 26 pg MeIQx/g), rat (133 +/- 15 pg/g) or mouse (78 +/- 10 pg/g) tissues; and no difference existed between the levels detected in human normal and tumor tissue (101 +/- 15 pg/g). Mean DNA-adduct levels in normal human colon (26 +/- 4 adducts/10(12) nucleotides) were significantly greater (p < 0.01) than in rats (17.1 +/- 1 adduct/10(12) nucleotides) or mice (20.6 +/- 0.9 adduct/10(12) nucleotides). No difference existed in adduct levels between normal and tumor tissue in humans. These results show that MeIQx forms DNA adducts in human colon at low dose, and that the human colon may be more sensitive to the effects of MeIQx than that of mice or rats.

Accelerator mass spectrometry (AMS) is a nuclear physics technique developed about twenty years ago, that uses the high energy (several MeV) of a tandem Van de Graaff accelerator to measure very small quantities of rare and long-lived isotopes. Elements that are of interest in biomedicine and environmental sciences can be measured, often to parts per quadrillion sensitivity, i.e. zeptomole to attomole levels (10(-21)-10(-18) mole) from milligram samples. This is several orders of magnitude lower than that achievable by conventional decay counting techniques, such as liquid scintillation counting (LSC). AMS was first applied to geochemical, climatological and archaeological areas, such as for radiocarbon dating (Shroud of Turin), but more recently this technology has been used for bioanalytical applications. In this sphere, most work has been conducted using aluminium, calcium and carbon isotopes. The latter is of special interest in drug metabolism studies, where a Phase 1 adsorption, distribution, metabolism and excretion (ADME) study can be conducted using only 10 nanoCurie (37 Bq or ca. 0.9 microSv) amounts or less of 14C-labelled drugs. In the UK, these amounts of radioactivity are below those necessary to request specific regulatory approval from the Department of Health's Administration of Radioactive Substances Advisory Committee (ARSAC), thus saving on valuable development time and resources. In addition, the disposal of these amounts is much less an environmental issue than that associated with microCurie quantities, which are currently used. Also, AMS should bring an opportunity to conduct "first into man" studies without the need for widespread use of animals. Centre for Biomedical Accelerator Mass Spectrometry (CBAMS) Ltd. is the first fully commercial company in the world to offer analytical services using AMS. With its high throughput and relatively low costs per sample analysis, AMS should be of great benefit to the pharmaceutical and biotechnology industries as well as other life science areas.